Mercury pollutant brings huge threat to human health and the environment because of its bioaccumulation and permanent damage. Thus, highly selective and ratiometric detection and imaging of Hg2+ in organisms is quite important. Herein, we report on a polymer nanoparticle-based ratiometric fluorescent probe (NP3) for highly selective recognition of Hg2+ in totally aqueous solution and imaging in living cells. This probe comprises the reference fluorescent dye (4-ethoxy-9-allyl-1,8-naphthalimide: EANI), which resides in the core of nanoparticle, and the Hg2+-recognition group (fluorescein derivative: AEMH-FITC) on the surface of nanoparticle. It displays distinct dual emissions at 432 nm and 528 nm under a single wavelength excitation. The fluorescence of the FITC moieties can be effectively quenched by Hg2+ due to photoinduced electron transfer (PET) mechanism, while that of the EANI remains constant, resulting in a ratiometric fluorescent detection of Hg2+. The as-prepared NP3 not only shows favorable water dispersibility, excellent long-term photostability, but also exhibits highly selective ratiometric detection of Hg2+ over other interfering ions (such as Cu2+, Na+, Mg2+, Zn2+, Co2+, Mn2+, K+, Ca2+, Fe3+, Cd2+, Ag+, Fe2+), and quite low detection limit (～75 nM). Furthermore, intracellular fluorescence imaging experiments demonstrate that NP3 has good permeability of cell-membrane, and is able to facilitate visualization of Hg2+ changes in living cells.